Method of fermentation

ABSTRACT

The invention provides a process for the production of biomass by culturing a microorganism in an aqueous liquid culture medium circulating in a loop reactor having an effluent gas removal zone where from carbon dioxide-containing effluent gas is removed from the reactor and upstream thereof a degassing zone in which a driving gas is introduced to drive carbon dioxide in the liquid phase into a separable effluent gas phase and having upstream of said degassing zone a nutrient gas introduction zone in which oxygen is introduced into the reactor and mixed with the liquid culture medium therein, characterised in that oxygen introduction in said nutrient gas introduction zone is effected at a plurality of locations along the flow path through said loop reactor at a rate such that the average dissolved oxygen content of said liquid culture medium measured using a polarographic oxygen electrode does not exceed 25 ppm.

[0001] This invention relates to improvements in and relating toprocesses for fermenting microorganisms, e.g. for the generation ofbiomass or the preparation of materials produced by microorganisms, andto fermentor apparatus for the performance thereof, in particularprocesses for culturing microorganisms in a loop reactor using a gaseousnutrient.

[0002] Recently, much attention has been directed toward the developmentof new sources of protein which may be incorporated into foods for humanand/or animal consumption. A number of different protein-containingmaterials have been proposed as substitutes for more traditional sourcesof protein, such as fish meal, soya products and blood plasma, in humanfoods and as animal feeds. These materials include single-cellmicroorganisms such as fungi, yeasts and bacteria which contain highproportions of proteins. These may be grown by reproduction producingbiomass through the growth of the microorganisms on hydrocarbon or othersubstrates. Today, the most widely used protein-containingmicroorganisms (also referred to herein as “single-cell proteins”) arethose derived from fungi or yeast.

[0003] Single-cell protein materials can be used directly in foods, e.g.as a spray dried product, or the biomass may be further processed, e.g.by hydrolysis and/or separation, before use.

[0004] Besides being simply used as sources of biomass, microorganismsmay be grown and harvested to serve as sources of useful chemicals, e.g.drug compounds, proteins, carotenoids, etc. Thus for example Du Pont inWO 02/18617, WO 02/20815, WO 02/20728 and WO 02/20733 (the contents ofwhich are hereby incorporated by reference) describe the use ofmethanotrophic bacteria, and in particular Methylomonas 16 a (ATCC PTA2402), for the preparation of carotenoids.

[0005] In DK-B-170824 (Dansk Bioprotein A/S) and in WO01/60974 (NorfermDA), the contents of both of which are hereby incorporated by reference,is described a loop reactor for use in cultivating microorganisms togenerate biomass, e.g. for culturing methanotrophic bacteria to generatea material which can for example be used in or as or as a precursor tohuman or animal feed.

[0006] The loop reactor described in DK-B-170824 has a top unitconnected to a conduit having, in order, a down-flow section, asubstantially horizontal flow section, an up-flow section and anon-vertical out-flow section. The top unit is cylindrical with its axisvertical and with the down-flow section of the conduit attached to itsbase. The out-flow section of the conduit extends tangentially from aside of the top unit. In this way, effluent gas and liquid culturemedium entering the top unit from the out-flow section may be separatedefficiently with the gas being withdrawn from the headspace of the topunit and the liquid being returned to the down-flow section of theconduit. The aqueous liquid culture medium is driven around the reactorloop, with gaseous and non-gaseous nutrients (e.g. oxygen, methane,nitrogen sources and minerals) being introduced into the conduit andmixed with the liquid culture medium therein, with biomass-containingculture medium being withdrawn from the conduit, and with effluent gas(e.g. carbon dioxide-containing gas) being withdrawn from the top unit.Nutrient addition and effluent gas withdrawal is generally continuous,while biomass withdrawal may be continuous or batchwise.

[0007] In the loop reactor described in DK-B-170824 the circulation rateof the reaction medium was generally about 30 to 90 seconds per circuit.Since unconsumed oxygen and methane in the effluent gas would make thispotentially explosive and would be uneconomical since oxygen and methanewould be wasted, gaseous nutrient addition could not occur at all pointsalong the conduit. Likewise, to ensure efficient separation of carbondioxide from the liquid reaction medium for venting from the top unit,gas/liquid mixing could not be effected at all points along the conduiteither. In practice therefore, in the loop reactor of DK-B-170824,gaseous nutrient addition was such that the dissolved oxygen content inthe culture medium would drop to about zero by the time the culturemedium reached the top of the upflow section of the conduit.

[0008] However, while the loop reactor described in DK-B-170824 can besuccessfully used for biomass production, when used at high biomassconcentrations (e.g. above 5 g/L) the production process is susceptibleto occasional sudden failure, requiring the process to be restarted withfresh live bacteria.

[0009] We have now found that this problem may be reduced or avoided ifnutrient gases (e.g. methane and/or oxygen, particularly oxygen) aresupplied into the conduit so as to reduce the time the culture medium isessentially free of an essential nutrient and/or so as to avoid orminimize periods of unduly high concentrations of nutrient gases in theculture medium.

[0010] Thus in one aspect the invention provides a process for theproduction of biomass by culturing a microorganism, preferably amethanotrophic bacterium, in an aqueous liquid culture mediumcirculating in a loop reactor having an effluent gas removal zonewherefrom carbon dioxide-containing effluent gas is removed from thereactor and upstream thereof a degassing zone in which a driving gas isintroduced to drive carbon dioxide in the liquid phase into a separableeffluent gas phase and having upstream of said degassing zone a nutrientgas introduction zone in which oxygen, and preferably also methane, areintroduced into the reactor and mixed with the liquid culture mediumtherein, characterised in that oxygen introduction in said nutrient gasintroduction zone is effected at a plurality of locations along the flowpath through said loop reactor at a rate such that the average dissolvedoxygen content of said liquid culture medium measured using apolarographic oxygen electrode (e.g. in mixer-free sections of thereactor) does not exceed 25 ppm, more preferably 20 ppm, especially 15ppm, more especially 10 ppm, and preferably also such that the dissolvedoxygen content of said liquid culture medium at each said location (withthe optional exception of the first said location downstream of saiddegassing zone) is at least 0.5 ppm, especially at least 1 ppm, moreespecially at least 3 ppm.

[0011] In this aspect of the invention, the oxygen introductionlocations are preferably so spaced that the flow time between locationsis less than 20 seconds, especially less than 16 seconds, particularlyless than 6 seconds, more preferably less than 5 seconds, still morepreferably less than 4 seconds, other than between the last and firstlocations (ie those immediately upstream and downstream of the degassingzone).

[0012] By average dissolved oxygen content is meant the average along adiameter of the cross sectional plane of the reactor. Where the reactoris not circular in cross section, the term diameter should be understoodto mean maximum dimension between inside walls. Such an average may betaken by measuring dissolved oxygen content at several, e.g. 3, 5 or 7,equally spaced points along the diameter.

[0013] The dissolved oxygen content should desirably be measured atmixer-free sections of the loop reactor, ie places within the reactorwhere the cross section through the reactor does not pass through amixer device. Desirably the dissolved oxygen content is measured atmixer-free sections downstream of the oxygen introduction zones and thesubsequent mixers.

[0014] Thus the dissolved oxygen content of the culture medium isconveniently measured using a polarographic oxygen electrode (availablecommercially, e.g. from Ingold) at positions between the oxygenintroduction locations sufficiently far downstream of the previous suchlocation that mixing of oxygen and culture medium is substantiallycomplete.

[0015] In order that oxygen concentration extremes may be avoided onoxygen introduction, introduction is preferably effected through aplurality of apertures at each introduction location, e.g. an array ofapertures spaced apart in the plane transverse to the flow direction andoptionally also along the flow direction. This may be achieved bydispersing in the reactor multi-armed, perforated gas distributors,preferably with the pressure drop from inside to outside the distributorbeing up to 6 bar, e.g. 0.4 to 3 bar especially 0.6 to 1 bar. Suchdistributors are conveniently disposed in the gaps between adjacentstatic mixer arrays in the reactor. In one preferred embodiment, gasdistributors are also disposed within the static mixer arrays, e.g. bydisposing perforated tubes along mixer channels or by the use ofperforated corrugated panels that function both as gas distributors andas plates within the mixer array. Hollow panel mixer plates throughwhich coolant (e.g. cold water) is flowed may also be used to cool theculture medium in the reactor.

[0016] The dissolved oxygen content is preferably measured upstream ofeach oxygen introduction location with the measured values being fed toa computer arranged to control nutrient introduction rates, cooling,biomass withdrawal, etc. In this way the dissolved oxygen content can bemaintained within the desired range throughout the reactor and forextended operation periods.

[0017] Desirably, the oxygen content of the culture medium in theeffluent gas removal zone, or at the lowest dissolved oxygenconcentration point in the loop, is less than 0.1 ppm by weight. Thisresults in the biomass product having greater bioavailability as afeedstuff.

[0018] While the dissolved oxygen content of the liquid culture mediummay drop to negligible or undetectable levels, i.e. about 0 ppm byweight, it is preferred that it should remain at detectable levels overat least a significant proportion of the path length through thereactor. Thus viewed from a further aspect the invention provides aprocess for the production of biomass by culturing a microorganism,preferably a methanotrophic bacterium, in an aqueous liquid culturemedium circulating in a loop reactor having an effluent gas removal zonewherefrom carbon dioxide-containing effluent gas is removed from thereactor and upstream thereof a degassing zone in which a driving gas isintroduced to drive carbon dioxide in the liquid phase into a separableeffluent gas phase and having upstream of said degassing zone a nutrientgas introduction zone in which oxygen, and preferably also methane, areintroduced into the reactor and mixed with the liquid culture mediumtherein, characterised in that oxygen introduction in said nutrient gasintroduction zone is such that between said nutrient gas introductionzone and said degassing zone the dissolved oxygen content of the liquidculture medium does not fall below 3 ppm by weight (preferably at least4 ppm, especially at least 5 ppm).

[0019] Viewed from a still further aspect the invention provides aprocess for the production of biomass by culturing a microorganism,preferably a methanotrophic bacterium, in an aqueous liquid culturemedium circulating in a loop reactor having an effluent gas removal zonewherefrom carbon dioxidè-containing effluent gas is removed from thereactor and upstream thereof a degassing zone in which a driving gas isintroduced to drive carbon dioxide in the liquid phase into a separableeffluent gas phase and having upstream of said degassing zone a nutrientgas introduction zone in which oxygen, and preferably also methane, areintroduced into the reactor and mixed with the liquid culture mediumtherein, characterised in that oxygen introduction into said liquidculture medium is so effected that the dissolved oxygen content of saidliquid culture medium does not fall below 3 ppm by weight (preferably atleast 4 ppm, especially at least 5 ppm) over a path length of the loopreactor corresponding to more than 30 seconds, preferably 20 seconds,more preferably 10 seconds, especially 5 seconds.

[0020] Viewed from a yet still further aspect the invention provides aprocess for the production of biomass by culturing a microorganism,preferably a methanotrophic bacterium, in an aqueous liquid culturemedium circulating in a loop reactor having an effluent gas removal zonewherefrom carbon dioxide-containing effluent gas is removed from thereactor and upstream thereof a degassing zone in which a driving gas isintroduced to drive carbon dioxide in the liquid phase into a separableeffluent gas phase and having upstream of said degassing zone a nutrientgas introduction zone in which oxygen, and preferably also methane, areintroduced into the reactor and mixed with the liquid culture mediumtherein, characterised in that oxygen introduction in said nutrient gasintroduction zone is such that between said nutrient gas introductionzone and said degassing zone the dissolved oxygen content of the liquidculture medium does not fall below X ppm by weight, where X is definedby

X=1.35 Y.B

[0021] where B is the biomass content of the culture medium in g/L and Yis from 0.75 to 1.25, preferably 0.80 to 1.20, especially 0.85 to 1.15,more preferably 0.90 to 1.10, more especially 0.95 to 1.05, and B isgreater than 5, especially greater than 10, particularly 15 to 30,especially 18 to 25.

[0022] Viewed from a still further aspect the invention provides aprocess for the production of biomass by culturing a microorganism,preferably a methanotrophic bacterium, in an aqueous liquid culturemedium circulating in a loop reactor having an effluent gas removal zonewherefrom carbon dioxide-containing effluent gas is removed from thereactor and upstream thereof a degassing zone in which a driving gas isintroduced to drive carbon dioxide in the liquid phase into a separableeffluent gas phase and having upstream of said degassing zone a nutrientgas introduction zone in which oxygen, and preferably also methane, areintroduced into the reactor and mixed with the liquid culture mediumtherein, characterised in that oxygen introduction in said nutrient gasintroduction zone is such that the dissolved oxygen content of theliquid culture medium over at least 30% (preferably at least 50%, morepreferably at least 60%) of the path length of the loop reactor is atleast 10 ppm by weight, the dissolved oxygen content of the liquidculture medium immediately prior to introduction of said driving gas insaid degassing zone is at least 3 ppm by weight (preferably at least 4ppm, especially at least 5 ppm), and the oxygen content of said effluentgas is at least 1 mole % (preferably at least 2 mole %, e.g. 2 to 8 mole%).

[0023] These methods described herein as further aspects of theinvention are preferably combined with the method of the invention asfirst defined.

[0024] In the process of the invention, it is preferred to drive theliquid culture medium around the loop reactor using a propeller. It isespecially preferred to use a propeller having overlapping or multiple,radially curved blades, i.e. a low-cavitation propeller. Suchlow-cavitation propellers are well-known in the field of submarinedesign. By overlapping blades it is meant that at leat one line existsthat is parallel to the propeller axis and that passes through at leasttwo blades. By a radially curved blade it is meant that there is anangle between the radial lines passing through the base and tip of theblade. The use of such propellers in a loop fermentation reactor isnovel and forms a further aspect of the invention. Thus viewed from afurther aspect the invention provides a process for the production ofbiomass by culturing a microorganism, preferably a methanotrophicbacterium, in an aqueous liquid culture medium circulating in a loopreactor (preferably one having an effluent gas removal zone wherefromcarbon dioxide-containing effluent gas is removed from the reactor andupstream thereof a degassing zone in which a driving gas is introducedto drive carbon dioxide in the liquid phase into a separable effluentgas phase and having upstream of said degassing zone a nutrient gasintroduction zone in which oxygen and preferably also methane areintroduced into the reactor and mixed with the liquid culture mediumtherein), characterised in that said liquid culture medium is circulatedthrough said loop reactor under the action of a propeller havingoverlapping or multiple, radially curved blades.

[0025] Using such low cavitation propellers, it is possible to pumpliquids having a greater gas content than with conventional propellersand thus it is possible to have nutrient gases introduced into theliquid culture medium closer upstream of the propeller than is otherwisepossible. In this way, the proportion of the reactor path length whichhas low dissolved gas content may be reduced.

[0026] The mixing of the nutrient gases into the liquid culture mediumis an important aspect of the performance of the process of theinvention. We have found that the use of static mixers, each comprisingan array of mixer plates, positioned downstream of nutrient gasinjection points results in a particularly efficient dissolution of thenutrient gases even though the gas:liquid volume ratio is relativelysmall. Such use of mixer plate arrays in loop fermentation reactors isnovel and forms a further aspect of the invention. Viewed from thisaspect the invention provides a process for the production of biomass byculturing a microorganism, preferably a methanotrophic bacterium, in anaqueous liquid culture medium circulating in a loop reactor (preferablyhaving an effluent gas removal zone wherefrom carbon dioxide-containingeffluent gas is removed from the reactor and upstream thereof adegassing zone in which a driving gas is introduced to drive carbondioxide in the liquid phase into a separable effluent gas phase andhaving upstream of said degassing zone a nutrient gas introduction zonein which oxygen and preferably also methane are introduced into thereactor and mixed with the liquid culture medium therein), characterisedin that nutrient gas and liquid, culture medium are mixed in said loopreactor by passage through a static mixer comprising a stack of parallelcorrugated flexible (and preferably perforated) plates arranged with thestacking direction perpendicular to the direction of flow of said fluidmedium and with the corrugation ridges thereof angled to said directionof flow (e.g. at an angle of 20 to 70°, preferably 40 to 50°, especially45°, relative to the direction of flow) and with their angle to thedirection of flow being substantially equal and opposite for adjacentplates.

[0027] The stacking direction, i.e. the normal to the surface of aplanar sheet placed over either major surface of a mixer plate, may beanywhere between vertical and horizontal. However the stacking directionbetween successive stacks is preferably rotated through 90°, morepreferably 80 to 90°, most preferably about 90°, 0°, 90°, 0° etc. or+45°, −45°, −45° relative to the vertical. Especially preferably thestacking direction for successive stacks is not 0°, 0°, 0° etc. relativeto the vertical.

[0028] In these last two aspects of the invention, indeed in mostaspects of the invention, the loop reactor is preferably one having acentre line, i.e. flow path length, of at least 40 m, more preferably atleast 80 m.

[0029] Viewed from a further aspect, the invention provides a processfor generating biomass by culturing a microorganism in a liquid reactionmedium circulating in a loop reactor having an effluent gas-liquidreaction medium separating zone upstream of an effluent gas removalzone, characterized in that oxygen and/or methane is fed into the liquidreaction medium in said separating zone.

[0030] Viewed from a further aspect, the invention provides a fermentorapparatus comprising a loop reactor comprising an effluent gas-liquidreaction medium separating zone upstream of an effluent gas removalzone, characterized in that said separating zone has an inlet forfeeding oxygen and/or methane into liquid reaction medium therein.

[0031] The loop reactor used in the processes of the present inventionpreferably is one which relies on propulsion rather than gas uplift tomove the liquid culture medium around the loop. Since it is easier tomaintain nutrient gas and liquid culture medium properly mixed in asubstantially horizontal section of the loop, and since mass transfer(i.e. transfer of nutrient gas into the liquid phase) increases withincreased pressure in the culture medium, the loop preferably comprisesa substantially vertical down-flow zone from the effluent gas removalzone followed by a substantially horizontal (e.g. “U” shaped) zone inturn followed by a substantially vertical up-flow zone leading back tothe effluent gas removal zone. In order that degassing and effluentgas/liquid culture medium should be particularly effective, between thesubstantially vertical up-flow zone and the effluent gas removal zone,it is desirable to have a substantially non-vertical, e.g. horizontal,out-flow zone between the two. This, in which most or all effluentgas/liquid culture medium separation occurs, may have a uniformgradient, may gradually become more horizontal or may change stepwisetowards horizontal. The driving gas, i.e. the gas used to displacecarbon dioxide from the dissolved phase (usually nitrogen but optionallyanother inert non-flammable gas) may for example be introduced at one ormore points from the beginning of the substantially vertical up-flowzone to the entry into the effluent gas removal zone, howeverparticularly preferably it will be introduced at one or more pointsbetween the upper portion (e.g. the upper 20%, more preferably the upper10%) of the vertical portion of the up-flow zone and the beginning ofthe flattest (i.e. most horizontal) portion of the out-flow zone.Especially preferably, the driving gas is introduced in the upperportion of the vertical portion of the up-flow zone. In this context itwill be understood that the “point” at which a gas may be introduced mayhave an extended length within the liquid path within the loop, e.g.where gas is introduced through a series of inlet ports or through amultiply perforated inlet port.

[0032] In general, where the centre line of the loop, outside the gaseffluent removal zone, changes direction, this will be by curvature ofthe loop rather than by a sharp angled bend so as to improve the flowproperties of the liquid culture medium.

[0033] The gas pressure in the headspace in the gas effluent removalzone will preferably be from −0.5 to +1.0 atmosphere relative to ambientpressure, especially +0.2 to +0.6. The vertical drop between thegas-liquid surface at the end of the outflow zone and the centre line ofthe loop in the horizontal zone is preferably at least 10 m, especiallyat least 18 m, e.g. 18 to 30 m.

[0034] The cross sectional area of the loop reactor (outside the gaseffluent removal zone) may be constant but preferably is increased atleast in the outflow zone, e.g. to a maximum value of at least 2.0times, more preferably at least 4.0 times that in the minimum crosssectional area part of the down flow and horizontal flow zones (whichmay typically be before the propeller). Typically, the inner diametersof the loop, outside the gas effluent removal zone may be in the range30 cm to 30 m, especially 1.0 to 2.5 m. Typically also the centre lineof the loop, excluding the gas effluent removal zone, has a length inthe range 40 to 200 m, preferably 80 to 150 m, preferably with at least50%, more preferably at least 60%, especially at least 70%, of this inthe horizontal zone. The outflow zone preferably has a centre linelength of 0.5 to 10 m, especially 1.5 to 8 m.

[0035] The loop reactor will generally be circular in cross-sectionoutside the gas removal zone; however in the separation zone otherconfigurations, e.g. rectangular, elliptical or ovoid cross-sections,may be adopted to enhance gas/liquid separation.

[0036] Nutrient gas introduction in the loop is preferably effected atleast three, more preferably at least six, positions along the length ofthe loop, preferably with at least 60% being introduced in thehorizontal zone. While it is preferred that nutrient gases be introducedin the downflow section, this may require the use of a low cavitationpropeller for subsequent propulsion of the liquid culture medium. Suchpropeller designs are well-known in the field of submarine construction.Typical low-cavitation propellers may have over-lapping blades, ormultiple (e.g. at least 6) radially-curved blades (i.e. blades where thebase and tip are radially displaced from each other). Unlike in priorreactor designs where nutrient gas input was designed to give asubstantially zero dissolved oxygen content in the culture medium by thebeginning of the degassing zone, it is desirable to introduce nutrientgases, in particular oxygen, in such amounts that dissolved oxygencontent at the beginning of the degassing zone, is at least 3 ppm. Tothis end, some of the nutrient gas, e.g. up to about 25% of the nutrientgas, is desirably introduced in the upflow zone.

[0037] As excess nutrient gas can give rise to toxicity problem for themicroorganisms being cultured in the reactor, oxygen introduction ispreferably such as to achieve a maximum dissolved oxygen content in theliquid culture medium of no more than 25 ppm, especially no more than 20ppm, more especially no more than 15 ppm. Likewise to maximise processefficiency in terms of biomass production, methane is preferablyintroduced in a 1:1to 1:3, especially 1:1.2 to 1:2.5, more especiallyabout 1:1.8, mole ratio relative to oxygen.

[0038] Methane may be used in purified form or in a gas mixture, e.g.natural gas or methane-enriched natural gas. Likewise purified oxygen oroxygen in a gas mixture (e.g. air or oxygen-enriched air) may be used.Where air is used, it is preferably filtered to avoid introduction oftoxic impurities.

[0039] The methane and oxygen used may likewise be in gaseous (e.g.compressed) or liquefied form; in the latter case however pre-heatingwill generally be required to prevent the nutrient gas cooling theliquid culture medium unduly.

[0040] Natural gas mainly consists of methane, although its compositionwill vary for different gas fields. Typically, natural gas may beexpected to contain about 90% methane, about 5% ethane, about 2% propaneand some higher hydrocarbons. During the fermentation of natural gas,methane is oxidized by methanotrophic bacteria to biomass and carbondioxide. Methanol, formaldehyde and formic acid are metabolicintermediates. Formaldehyde and to some extent carbon dioxide areassimilated into biomass. However, methanotrophic bacteria are unable touse substrates comprising carbon-carbon bonds for growth and theremaining components of natural gas, i.e. ethane, propane and to someextent higher hydrocarbons, are oxidized by methanotrophic bacteria toproduce the corresponding carboxylic acids (e.g. ethane is oxidized toacetic acid). Such products can be inhibitory to methanotrophic bacteriaand it is therefore important that their concentrations remain low,preferably below 50 mg/l, during the production of the biomass. Onesolution to this problem is the combined use of one or moreheterotrophic bacteria which are able to utilize the metabolitesproduced by the methanotrophic bacteria. Such bacteria are also capableof utilizing organic material released to the fermentation broth by celllysis. This is important in order to avoid foam formation and alsoserves to minimize the risk of the culture being contaminated withundesirable bacteria. A combination of methanotrophic and heterotrophicbacteria results in a stable and high yielding culture.

[0041] Besides oxygen and methane, other nutrients, e.g. minerals and anitrogen source (e.g. ammonia, nitrates, urea, etc.) will generally beadded to the liquid culture medium. Unlike oxygen and methane howeverthe degassing operation does not critically affect their concentrationand thus their addition can generally each be at only one, two or threepoints along the loop. For certain minerals, in particular copper, itmay however be desirable to effect introduction at a higher number ofpoints along the loop. For copper this is relevant as increased copperconcentration serves to increase methane consumption.

[0042] Air or pure oxygen may be used for oxygenation and ammonia ispreferably used as the nitrogen source. In addition to these nutrients,the bacterial culture will typically require water, phosphate (e.g. asphosphoric acid) and several minerals which may include magnesium,calcium, potassium, iron, copper, zinc, manganese, nickel, cobalt andmolybdenum, typically used as sulphates, chlorides or nitrates. Allminerals used in the production of the single-cell material should be offood-grade quality.

[0043] In the process of the invention it is desirable to monitor carbondioxide, oxygen and methane content in the effluent gas, and thebiomass, nitrogen, dissolved oxygen, phosphate and mineral contents ofthe culture medium. Biomass content may be measured using samples of thebiomass-containing medium extracted for further processing; e.g. byseparating biomass from liquid by centrifugation and weighing. Nitrogen,phosphate and mineral contents may also be measured in this extractedmaterial, e.g. using standard procedures, e.g. atomic absorption, etc.Dissolved oxygen content, again measured by standard procedures, ispreferably monitored at two or more points about the loop. Effluent gasis preferably sampled and cooled to about 5° C. whereafter oxygen ispreferably determined by measuring the paramagnetism of the gas andmethane and carbon dioxide by infra-red spectrometry. Typically oxygencontent is preferably about 7.5% (v/v), methane content about 4.0% (v/v)and carbon dioxide content about 35% (v/v) when the process is runningsmoothly.

[0044] Ammonia concentration is preferably up to 200 ppm, especially 0.1to 5 ppm by weight. Biomass concentration is preferably up to 30 g/L,e.g. 5 to 20 g/L.

[0045] Phosphate content in the culture medium is preferably at least 10ppm by weight so as to minimize foaming at the top of the reactor,especially about 100 to 200 ppm.

[0046] Potassium, magnesium and calcium contents in the culture mediumare preferably at least 5, 0.5 and 0.5 ppm by weight, especially about100-200, 20-50 and 20-50 ppm respectively. Copper and iron contents mayconveniently be measured in the extracted biomass; preferably theirminimum contents are 5 and 200 mg/kg respectively.

[0047] During production of the single-cell material, the pH of thefermentation mixture will generally be regulated to between about 5.5and 7.5, e.g. to 6.5±0.3. Suitable acids/bases for pH regulation may bereadily selected by those skilled in the art. Particularly suitable foruse in this regard are sodium hydroxide and sulphuric acid. Duringfermentation the temperature within the fermentor should preferably bemaintained to within the range of from 40° C. to 50° C., most preferably45° C. ±2° C.

[0048] In operation of the process of the invention, liquid (e.g. someor all of the liquid nutrients, the liquid added to compensate forbiomass removal, a fraction of the liquid withdrawn from the reactor,cooled and returned to the reactor to control the temperature, etc.),optionally containing antifoam, is preferably sprayed onto the surfaceof the liquid culture medium in the degassing zone to reduce foam buildup. Likewise, the horizontal flow section of the degassing zone ispreferably provided in its upper section with transversely extendingbaffles to combat foam build up. As a further antifoaming measure, thereactor can be provided in the degassing zone with steam inlets arrangedto inject steam into the headspace above the liquid surface.

[0049] Any single-cell protein material may be produced in accordancewith the processes of the invention. However, preferred microorganismsinclude bacteria and yeasts. Any bacteria or yeast approved for use infood products may be used and suitable species may be readily selectedby those skilled in the art. Particularly preferably, the single-cellsfor use in the invention will be a microbial culture which consists ofmethanotrophic bacteria optionally in combination with one or morespecies of heterotrophic bacteria, especially preferably a combinationof methanotrophic and heterotrophic bacteria. As used herein, the term“methanotrophic” encompasses any bacterium which utilizes methane ormethanol for growth. The term “heterotrophic” is used for bacteria thatutilize organic substrates other than methane or methanol for growth.

[0050] While the process of the invention is especially suited to theproduction of biomass which can be used with relatively little furtherprocessing as a feedstuff or food additive, the process may also be usedfor the preparation of specific chemicals which are generated by themicroorganism(s) in the liquid culture medium. In this event, thepost-fermentation treatment of the culture medium will involveseparation out of the particular chemicals of interest, e.g. byconventional chemical techniques, optionally following lysis of themicroorganism cells to release the chemicals of interest. In thisembodiment of the invention, microorganisms which naturally produce thechemicals of interest (e.g. proteins, drugs, carotenoids, etc) or whichhave been genetically modified to produce the chemicals of interest, maybe used. Many such microorganisms are known from the literature; howeverit is particularly preferred to use methanotrophic bacteria.

[0051] Preferred bacteria for use in the invention include Methylococcuscapsulatus (Bath), a thermophilic bacterium originally isolated from thehot springs in Bath, England and deposited as NCIMB 11132 at TheNational Collections of Industrial and Marine Bacteria, Aberdeen,Scotland. M. capsulatus (Bath) has optimum growth at about 45° C.,although growth can occur between 37° C. and 52° C. It is agram-negative, non-motile spherical cell, usually occurring in pairs.The intracellular membranes are arranged as bundles of vesicular discscharacteristic of Type I methanotrophs. M. capsulatus (Bath) isgenetically a very stable organism without known plasmids. It canutilize methane or methanol for growth and ammonia, nitrate or molecularnitrogen as a source of nitrogen for protein synthesis.

[0052] Other bacteria suitable for use in the invention include theheterotrophic bacteria Ralstonia sp. (formerly Alcaligenes acidovorans)DB3 (strain NCIMB 13287), Brevibacillus agri (formerly Bacillus firmus)DB5 (strain NCIMB 13289) and Aneurinibacillus sp. (formerly Bacillusbrevis) DB4 (strain NCIMB 13288) which each have optimum growth at atemperature of about 45° C.

[0053]Ralstonia sp. DB3 is a gram-negative, aerobic, motile rodbelonging to the family Pseudomonadaceae which can use ethanol, acetate,propionate and butyrate for growth. Aneurinibacillus sp. DB4 is agram-negative, endospore-forming, aerobic rod belonging to the genusBacillus which can utilize acetate, D-fructose, D-mannose, ribose andD-tagatose. Brevibacillus agri DB5 is a gram-negative,endospore-forming, motile, aerobic rod of the genus Bacillus which canutilize acetate, N-acetyl-glucosamine, citrate, gluconate, D-glucose,glycerol and mannitol.

[0054] Especially preferred for use in the invention is a microbialculture comprising a combination of the methanotrophic bacteriumMethylococcus capsulatus (Bath) (strain NCIMB 11132), and theheterotrophic bacteria Ralstonia sp. DB3 (strain NCIMB 13287) andBrevibacillus agri DB5 (strain NCIMB 13289), optionally in combinationwith Aneurinibacillus sp. DB4 (strain NCIMB 13288). The role ofRalstonia sp. DB3 is to utilize acetate and propionate produced by M.capsulatus (Bath) from ethane and propane in the natural gas. Ralstoniasp. DB3 may account for up to 10%, e.g. about 6 to 8%, of the total cellcount of the resulting biomass. The role of Aneurinibacillus sp. DB4 andBrevibacillus agri DB5 is to utilize lysis products and metabolites inthe medium. Typically, Aneurinibacillus sp. DB4 and Brevibacillus agriDB5 will each account for less than 1% of the cell count duringcontinuous fermentation.

[0055] Suitable yeasts for use in the processes of the invention may beselected from the group consisting of Saccharomyces and Candida.

[0056] If desired, the process of the invention may be performed usingbacteria (or yeasts) genetically modified so as to generate a desiredchemical compound which can then be extracted from the intercellularfluid or the biomass harvested from the reactor. The scientific andpatent literature contains numerous examples of such geneticallymodified microorganisms including, inter alia, methanotrophic bacteria.

[0057] In one especially preferred embodiment of the invention, theprocess is performed using methanotrophic bacteria of the type describedin WO 02/18617 to produce carotenoids, e.g. antheraxanthin,adonixanthin, astaxanthin, canthaxanthin, zeaxanthin and the othercarotenoids mentioned on pages 39 and 40 of WO 02/18617. To this end,the methanotrophic bacterium Methylomonas 16a (ATCC PTA 2402) mayparticularly suitably be used. Carotenoids produced in this way may beseparated out from the liquid culture medium as described in WO02/18617, WO 02/20728 and WO 02/20733.

[0058] Ideally, the biomass produced from fermentation of natural gaswill comprise from 60 to 80% by weight crude protein; from 5 to 20% byweight crude fat; from 3 to 15% by weight ash; from 3 to 15% by weightnucleic acids (RNA and DNA); from 10 to 30 g/kg phosphorus; up to 500mg/kg iron; and up to 250 mg/kg copper. Particularly preferably, thebiomass will comprise from 68 to 73%, e.g. about 70% by weight crudeprotein; from 9 to 11%, e.g. about 10% by weight crude fat; from 5 to10%, e.g. about 7% by weight ash; from 8 to 12%, e.g. about 10% byweight nucleic acids (RNA and DNA); from 10 to 25 g/kg phosphorus; up to310 mg/kg iron; and up to 110 mg/kg copper. The amino acid profile ofthe protein content should be nutritionally favourable with a highproportion of the more important amino acids cysteine, methionine,threonine, lysine, tryptophan and arginine. Typically these may bepresent in amounts of about 0.7%, 3.1%, 5.2%, 7.2%, 2.5% and 6.9%,respectively (expressed as a per cent of the total amount of aminoacids). Generally the fatty acids will comprise mainly the saturatedpalmitic acid (approx. 50%) and the monounsaturated palmitoleic acid(approx. 36%). The mineral content of the product will typicallycomprise high amounts of phosphorus (about 1.5% by weight), potassium(about 0.8% by weight) and magnesium (about 0.2% by weight).

[0059] The biomass product of the invention is especially useful as acomponent or precursor in food products, particularly when used as asubstitute for natural plasma in animal feeds and in pet foods. Whenused in pet foods, additional ingredients may be added to the productsuch as fats, sugars, salt, flavourings, minerals, etc. The product maythen be formed into chunks resembling natural meat chunks in appearanceand texture. The product of the invention has the further advantagesthat this is readily formulated to contain necessary nutrients, iseasily digested by the animals and is palatable to the animals.

[0060] The product of the invention may find further use as a texturantin meat products (e.g. meat balls), as a replacement for plasma proteinsconventionally used as extenders in fresh meat to increase weight andvolume, as an emulsifier (e.g. in dressings, etc.), and in bakeryproducts to enhance dough properties.

[0061] When used in food products, the biomass, or processed biomassmaterial will typically be used in an amount of from 1 to 10% by weight,preferably up to 5% by weight. The exact proportion will depend on thedesired function of the material and can be readily determined by thoseskilled in the art. Typically, when used as a gelling agent this may bepresent in an amount of up to 20% by weight, e.g. 5 to 10% by weight(based on dry matter content of the product).

[0062] When the reactor was run without meeting the dissolved oxygencontent conditions specified above (in particular when overly highdissolved oxygen contents occurred), and when a certain biomass contentwas reached, although the reactor continued to perform satisfactorilyfor many hours, relatively suddenly ammonia usage dropped and within aperiod of only two or three hours the live bacterial population (andhence the biomass content of the material, if it continued to beextracted from the reactor) dropped to virtually zero. While in somecases, immediate cessation of ammonia feed, reduction or cessation ofbiomass extraction from the reactor and increase or decrease of oxygenfeed could “cure” the microorganism population and restore the biomassyield, in many cases this was not sufficient. Investigation of the deadbacteria showed that while these had not lysed the internal organellestructure had been significantly disrupted. This indicated that thebacterial genome was host to hitherto unrecognised viral nucleic acidsequences which under the extraordinary conditions experienced in thereactor were activated to express products which served to shut downnormal operation of the cell. It is believed that such genomic prophagerather than plasmid viral infection of monocellular microorganisms hashitherto not been identified and may likewise prove to be problematic inhigh biomass operation of aerobic loop reactor fermentors, e.g. withbacteria, fungi (e.g. fusarium), yeast or genetically modified cells(e.g. bacterial, mammalian (e.g: human, hamster, mouse, etc.) and it isa further aspect of the invention to use the solutions of the inventionrelating to the reduction of the dissolved oxygen depleted path lengthof the reactor in aerobic loop reactor fermentations of bacteria, fungi,yeast or genetically modified cells, e.g. in the production of alcohols(especially methanol or ethanol), foods or food additives or precursorstherefor, pharmaceuticals, antibodies, etc.

[0063] The rearrangement of the microorganism organelles observed onprocess malfunction may also be used as a control parameter for feedbackcontrol of fermentation reactors in general, and loop reactors inparticular, e.g. with observation of organelle rearrangement at all, orabove a threshold level, serving to trigger increase in nutrient supply,in particular oxygen and/or methane supply, or to reduce or halt ammoniasupply, etc. Such observation could for example be made using flowcytometry on cells removed from an extraction or sampling port in thereactor. This form of feedback control of fermentation reactors forms afurther aspect of the invention.

[0064] A primary indicator of malfunction in the operation of theprocesses of the invention is when a depressed pH in the culture mediumoccurs which recurs despite pH adjustment by base (e.g. NaOH) addition.This appears to be due to formic acid build up or inadequate formic acidto carbon dioxide conversion by the methanotrophic bacteria. An advancewarning of this problem could be obtained by monitoring theintracellular formic acid concentration of the bacteria, e.g. bysampling the culture medium, lysing the cells and spectrometricallyassaying for formic acid. Where the detected formic acid concentrationrises above a preset threshold, preventative action may be taken, e.g.by reducing oxygen and/or ammonia feed rates, etc. Besides intracellularformic acid, extracellular formic acid concentration may be monitoredand used as a process control parameter. Likewise dissolved oxygenconcentration, dissolved methane concentration and dissolved ammoniaconcentration may be monitored and used as a process control parameters.Desirably such monitoring is performed on line, i.e. at the reactor, andconventional monitoring apparatus (e.g. spectrometers and the like) maybe used. Such monitoring and control forms a further aspect of theinvention.

[0065] Viewed from a further aspect the invention provides afermentation reactor adapted for use in one or more processes accordingto the invention, e.g. by the inclusion of appropriately positionednutrient gas inlets, low cavitation propellers, stacked plate staticmixers, etc.

[0066] Viewed from a yet further aspect the invention providesbiologically generated products (e.g. biomass, drugs, antibodies,alcohols, etc.) produced by the processes of the invention and theiruses, e.g. as, in or as precursors for foodstuffs, as pharmaceuticals,as chemical raw materials, etc.

[0067] Where a loop reactor is used for cell culturing, the removal ofdissolved carbon dioxide from the culture medium is important andnitrogen is particularly suitable for use in this regard (i.e. as adriving gas as mentioned above).

[0068] As a result it is especially beneficial to locate the reactor inthe vicinity of (e.g. within 1 km of) an apparatus with a high demandfor oxygen, e.g. an alcohol or ethanol production plant, in this way thenitrogen produced in air separation (e.g. liquefication) to produceoxygen for one plant may be used in the fermentor apparatus, together ifdesired with a small proportion of the oxygen (e.g. as a nutrient gas inthe fermentor). Moreover, where the fermentor product is to be driedand, especially where it is oxygen sensitive on drying as is the casewith biomass production for bioproteins, the nitrogen may also be usedas the drying gas in the spray drier. Such synergistic co-locations ofnitrogen/oxygen separator, fermentor and oxygen-demanding plants form afurther aspect of the invention.

[0069] Where larger scale operation of the process of the invention isdesired, it is of course possible simply to increase the dimensions ofthe reactor and the power of the drive motor. An alternative however isto operate a set of reactors, e.g. arranged radiating out from a centralunit housing the degassing zones for all the reactors. One advantage ofusing a set (e.g. 2, 3, 4 or 5) of reactors is that reaction start-up inone reactor may be effected by inoculation with liquid culture mediumfrom a reactor which is already in operation. Since the reactorsgenerally do have to have down time for cleaning or repair, such anarrangement ensures that operation can be continuous and that thestart-up time for the individual reactors may be significantly reduced.

[0070] Embodiments of the processes and apparatus of the invention willnow be described further with reference. to the accompanying drawings,in which:

[0071]FIG. 1 is a schematic drawing of a loop reactor according to theinvention;

[0072]FIG. 2 is a cross section through a horizonal section of thereactor of FIG. 1 showing schematically the arrangement of the plates ofthe static mixer therein;

[0073]FIG. 3 is a schematic view of a further loop reactor according tothe invention; and

[0074]FIG. 4 is a cross section through a horizontal section of thereactor of FIG. 1 or FIG. 3 showing schematically the arrangement of theplates of the static mixer therein.

[0075] Referring to FIG. 1 there is a shown a loop reactor 1, the loopwhereof comprises a top unit 2 for effluent gas removal, a verticaldown-flow section 3, a horizontal flow section 4, a vertical up-flowsection 5, and a horizontal effluent gas/liquid reaction mediumseparation section 6. Down-flow section 3 is attached to the conicalbase of cylindrical top unit 2 while separation section 6 enters topunit 2 tangentially at a side so as to achieve cyclonic gas/liquidseparation. From the top of top unit 2, effluent gas is removed throughoutlet port 7. The top of the top unit is also provided with emergencyvent 8 which opens automatically if the effluent gas pressure in the topunit exceeds a pre-set maximum, e.g. 0.5 atmosphere above ambient.

[0076] The liquid culture medium 9 is circulated round the loop bypropeller 10 driven by motor 11 (e.g. a 650 kW motor). Upstream ofpropeller 10 is exit port 12 from which biomass is removed fordewatering and further processing, e.g. hydrolysis, spray drying etc.

[0077] Within the horizontal section 4 of the loop are disposed a seriesof nutrient gas (e.g. O₂ and CH₄) inlets 13 (e.g. diffuser plates) andstatic mixers 14. Nutrient gas inlets 15 and 16 are preferably alsoprovided in the downflow and upflow sections 3 and S. Ammonia andmineral inlets 17 and 18 and sampling ports are preferably providedaround the loop. Monitoring and sampling ports (not shown) are alsopreferably provided around the loop and in the head space of top unit 2.

[0078] Towards the top of upflow section 5 is provided a drive gas (e.g.nitrogen) inlet 19, for example a diffuser plate.

[0079] In the base of separation section 6, there is preferably provideda porous diffuser 20 for nutrient gas (especially oxygen) which iselongated along the flow direction so as to supply nutrient gas into theliquid separated out in this section.

[0080] Referring to FIG. 2, the loop reactor is shown containing a stackof parallel horizontal corrugated perforated plates 21 disposed oneabove the other with the corrugations in registry but alternating indirection so as to provide flow channels 22 for the culture medium.

[0081] Referring to FIG. 3 there is a shown a loop reactor 1, the loopwhereof comprises a top unit 2 for effluent gas removal, a verticaldown-flow section 3, a horizontal flow section 4, a vertical up-flowsection 5, and a horizontal effluent gas/liquid reaction mediumseparation section 6. Down-flow section 3 is attached to the conicalbase of cylindrical top unit 2 while separation section 6 enters topunit 2 at a side. From the top of top unit 2, effluent gas is removedthrough outlet port 7. The top of the top unit is also provided withemergency vent which opens automatically if the effluent gas pressure inthe top unit exceeds a pre-set maximum, e.g. 0.5 atmosphere aboveambient.

[0082] The liquid culture medium 9 is circulated round the loop by apropeller driven by a vertically dispersed drive shaft driven by motor(e.g. a 650 kW motor). Upstream of a propeller driven by a verticallydisposed drive shaft is exit port 12 from which biomass is removed fordewatering and further processing, e.g. hydrolysis, spray drying etc.

[0083] Within the horizontal section 4 of the loop are disposed a seriesof nutrient gas (e.g. O₂ and CH₄) inlets 13 (e.g. diffuser plates) andstatic mixers. Ammonia and mineral inlets 17 and 18 and sampling portsare preferably provided around the loop. Monitoring and sampling ports(not shown) are also preferably provided around the loop and in the headspace of top unit 2.

[0084] Towards the top of upflow section 5 is provided a drive gas (e.g.nitrogen) inlet 19, for example a diffuser plate.

[0085] Dissolved oxygen content is measured by probes 23 within thereactor. Ammonia concentration and cell density are measured by probes24 and 25 in the harvesting outlet to the reactor, gas composition (CO₂,O₂, CH₄) is measured by probe 26 in the off gas venting line.Temperature and pH are measured within the reactor by probes 27 and 28.The information from these probes is used as a feedback control wherebya yield-optimum gas and ammonia distribution is calculated based on amechanistic model of the fermentation process providing, repeatedly,optimal settings for the O₂, CH₄ and NH₃ introduction valves, for thediverted flow of culture medium from the reactor into heat exchangersand back into the reactor, and for acid/base dosing to optimise pH.

[0086] In FIG. 4 a more preferred mixer array is shown. In thisembodiment, the parallel vertical corrugated perforated plates 21 aredisposed with their corrugations angled with respect to the flowdirection and alternating in orientation.

[0087] The mixer plates are preferably flexible as in this way they areself-cleaning. To this end they are conveniently formed from stainlesssteel of at leat 0.2 mm thickness, e.g. about 0.5 to 1.5 mm, especially0.8 to 1.2 mm thickness. The maximum interplate spacing, i.e. thechannel height between corrugations, is preferably at least 25 mm, e.g.50 to 250 mm, more preferably 80 to 150 mm, especially 90 to 110 mm.

[0088] The following non-limiting Example serves further to illustratethe invention.

EXAMPLE 1 Preparation of Homogenized Biomass

[0089] A microbial culture comprising Methylococcus capsulatus (Bath)(strain NCIMB 11132), Ralstonia sp. (formerly Alcaligenes acidovorans)DB3 (strain NCIMB 13287) and Brevibacillus agri (formerly Bacillusfirmus) DBS (strain NCIMB 13289), and optionally and preferablyAneurinibacillus sp. DB4 (strain NCIMB 13288) is produced in a loop-typefermentor by continuous aerobic fermentation of natural gas in anammonium/mineral salts medium (AMS) at 45° C., pH 6.5. The AMS mediumcontains the following per litre: 10 mg NH₃, 75 Mg H₃PO₄, 380 mgMgSO₄.7H₂O, 100 mg CaCl₂.2H₂O, 200 mg K₂SO₄, 75 mg FeSO₄.7H₂O, 1.0 mgCuSO₄.5H₂O, 0.96 mg ZnSO₄.7H₂O, 120 μg CoCl₂.6H₂O, 48 μg MnCl₂.4H₂O, 36μg H₃BO₃, 24 μg NiCl₂.6H₂O and 1.20 μg NaMoO₄.2H₂O.

[0090] The fermentor is filled with water which has been heat-sterilizedat 125° C. for 10 secs. Addition of the different nutrients is regulatedaccording to their consumption. With gradual build-up over time,continuous fermentation is operated with 1-3% biomass (on a dry weightbasis).

[0091] The biomass is subjected to centrifugation in an industrialcontinuous centrifuge at 3,000 rpm, optionally followed byhomogenization in an industrial homogenizer (pressure drop: 1000 bar(100 MPa); inlet temperature: 15° C. to produce a homogenized biomass),followed by ultrafiltration using membranes having an exclusion size of200,000 Daltons.

1. A process for the production of biomass by culturing a microorganism in an aqueous liquid culture medium circulating in a loop reactor having an effluent gas removal zone where from carbon dioxide-containing effluent gas is removed from the reactor and upstream thereof a degassing zone in which a driving gas is introduced to drive carbon dioxide in the liquid phase into a separable effluent gas phase and having upstream of said degassing zone a nutrient gas introduction zone in which oxygen is introduced into the reactor and mixed with the liquid culture medium therein, characterised in that oxygen introduction in said nutrient gas introduction zone is effected at a plurality of locations along the flow path through said loop reactor at a rate such that the average dissolved oxygen content of said liquid culture medium measured using a polarographic oxygen electrode does not exceed 25 ppm.
 2. A process as claimed in claim 1, wherein said liquid culture medium contains a methanotrophic bacterium and wherein oxygen and methane are introduced into said reactor and mixed with said liquid culture medium.
 3. A process as claimed in claim 1, further comprising harvesting biomass from said reactor and optionally processing the harvested biomass.
 4. A process as claimed in claim 1, further comprising harvesting biomass containing liquid culture medium from said reactor and separating therefrom a chemical compound produced by said microorganism.
 5. A process as claimed in claim 1, wherein oxygen introduction in said nutrient gas introduction zone is effected at a plurality of locations along the flow path through said loop reactor at a rate such that the average dissolved oxygen content of said liquid culture medium measured using a polarographic oxygen electrode does not exceed 15 ppm.
 6. A process as claimed in claim 1, wherein oxygen introduction in said nutrient gas introduction zone is effected at a plurality of locations along the flow path through said loop reactor at a rate such that the dissolved oxygen content of said liquid culture medium at each said location (with the optional exception of the first said location downstream of said degassing zone) is at least 0.5 ppm.
 7. A process as claimed in claim 1, wherein oxygen introduction in said nutrient gas introduction zone is such that the dissolved oxygen content of the liquid culture medium over at least 30% of the path length of the loop reactor is at least 10 ppm by weight, the dissolved oxygen content of the liquid culture medium immediately prior to introduction of said driving gas in said degassing zone is at least 3 ppm by weight, and the oxygen content of said effluent gas is at least 1 mole %.
 8. A process as claimed in claim 1, wherein oxygen introduction in said nutrient gas introduction zone is such that between said nutrient gas introduction zone and said degassing zone the dissolved oxygen content of the liquid culture medium does not fall below 3 ppm by weight.
 9. A process as claimed in claim 1, wherein oxygen introduction into said liquid culture medium is so effected that the dissolved oxygen content of said liquid culture medium does not fall below 3 ppm by weight over a path length of the loop reactor corresponding to more than 30 seconds.
 10. A process as claimed in claim 1, wherein said fluid culture medium is circulated through said loop reactor under the action of a propeller having overlapping or multiple, radially curved blades.
 11. A process as claimed in claim 1, wherein nutrient gas and liquid culture medium are mixed in said loop reactor by passage through a static mixer comprising a stack of parallel corrugated flexible plates arranged with the stacking direction perpendicular to the direction of flow of said fluid medium and with the corrugation ridges thereof angled to said direction of flow and with their angle to the direction of flow being substantially equal and opposite for adjacent plates.
 12. A process as claimed in claim 1, wherein oxygen introduction in said nutrient gas introduction zone is such that between said nutrient gas introduction zone and said degassing zone the dissolved oxygen content of the liquid culture medium does not fall below X ppm by weight, where X is defined by X=1.35 Y.Bwhere B is the biomass content of the culture medium in g/L and Y is from 0.75 to 1.25, and B is greater than
 5. 13. A process for generating biomass by culturing a microorganism in a liquid reaction medium circulating in a loop reactor having an effluent gas-liquid reaction medium separating zone upstream of an effluent gas removal zone, characterized in that oxygen and/or methane is fed into the liquid reaction medium in said separating zone.
 14. A process for the production of biomass by culturing a microorganism in an aqueous liquid culture medium circulating in a loop reactor, characterised in that said liquid culture medium is circulated through said loop reactor under the action of a propeller having overlapping or multiple, radially curved blades.
 15. A process for the production of biomass by culturing a microorganism in an aqueous liquid culture medium circulating in a loop reactor, characterised in that nutrient gas and liquid culture medium are mixed in said loop reactor by passage through a static mixer comprising a stack of parallel corrugated flexible plates arranged with the stacking direction perpendicular to the direction of flow of said fluid medium and with the corrugation ridges thereof angled to said direction of flow and with their angle to the direction of flow being substantially equal and opposite for adjacent plates.
 16. A process as claimed in claim 1, wherein said loop reactor has a flow path of at least 40 m.
 17. A fermentation reactor adapted for use in a process as claimed in claim
 1. 18. A fermentor apparatus comprising a loop reactor comprising an effluent gas-liquid reaction medium separating zone upstream of an effluent gas removal zone, characterized in that said separating zone has an inlet for feeding oxygen and/or methane into liquid reaction medium therein.
 19. A fermentor apparatus comprising a loop reactor containing a static mixer comprising a stack of parallel corrugated flexible plates arranged with the stacking direction perpendicular to the direction of flow in said reactor and with the corrugation ridges thereof angled to said direction of flow and with their angle to the direction of flow being substantially equal and opposite for adjacent plates.
 20. A fermentor apparatus comprising a loop reactor containing a propeller having overlapping or multiple, radially curved blades. 